Ο ρόλος και οι εφαρμογές

των οξυγαλακτικών βακτηρίων

στα τρόφιμα και την υγεία

(ΙΙ)

ΜΕΤΑΒΟΛΙΣΜΟΣ

Dairy Products

Mean composition of bovine milk

Water 86.6

Fat 4.1 2000-6000 (globules)

1010

Proteins- caseins- whey

3.650-300 (micelles)

4-6 (molecules)1014

1017

Lactose 5.0 0.5 (molecules) 1019

composition(%)

size(diameter, nm)

number / ml

Ash 0.7

Dairy Products

Fermented dairy products are known since ancient times

Fermentation contributes to

the preservation of vast quantities of nutritious foods

without significant fuel requirements

providing a wide diversity of

flavors

aromas

textures, but also,

enriches food with vitamins and essential amino acids

Dairy Products

Lactic acid bacteria convert fermentable sugars mainly to lactic acid,

and are thus responsible for processing and preserving milk

Other anti-microbial factors associated with lactic acid bacteria,

i.e. carbon dioxide

hydrogen peroxide

ethanol

diacetyl

bacteriocins

are responsible for inhibition of food spoilage and pathogens

Cheese

Cheese is one of the most diverse food groups

Around 500 varieties are produced from cow’s milk, while 500 are produced

from sheep’s and/or goat’s milk

Most cheeses are produced using as coagulant animal rennet

Several cheeses in the Iberian Peninsula are produced with the use of plant

rennet (an aqueous extract from the cardoon flowers; Cyanara cadunuculus)

Cheese

Smear surface-ripened

(Beaufort, Comte, Gruyere, Saint Paulin, Munster)

Mould surface-ripened

(Camembert, Brie, Roquefort, Gorgonzolla)

Swiss type

(Emmental, Gruyere, Beaufort, Graviera)

Cheese

Grana

(Parmigiano-Regiano, Grana Padano)

Pasta Filata

(Mozzarella, Provolone, Kashkaval, Kasseri)

White-brined

(Feta, Domiati, Beyaz peynir, Halloumi)

Whey cheeses

(Ricotta, Mizithra, Manouri, Anari, Karichee)

Fermented milks

They can be divided in 3 main classes based on the metabolic products

i) lactic fermentations (e.g. Yoghurt, Laban, Bulgarian buttermilk)

ii) yeast-lactic fermentations (Kefir, Koumiss)

iii) mould-lactic fermentations (Villi)

Closely related products are manufactured from fermented milks by

a) de-wheying to concentrate the product, which could resemble soft

cheese (e.g. Labneh)

b) drying of cereal/fermented milk mixture (e.g. Kishk or Trahana)

c) freezing fermented milk to resemble ice cream

Biochemical pathways leading to the formation of flavor compounds

The gray surface indicated compounds with a flavor note

Biochemistry of the Fermentation Process

Biochemistry of the Fermentation Process

The first essential step in milk fermentations is the catabolism

of milk lactose by the lactic acid bacteria (LAB)

The main end product is lactic acid (>50% of sugar carbon)

Lactic acid bacteria as a group exhibit an enormous capacity to

degrade different carbohydrates and related compounds

LAB adapt to various conditions and change their metabolism

accordingly, yielding thus significantly different end product patterns

Biochemistry of the Fermentation Process

Lactose is taken up via the PTS and enters the cytoplasm as lactose-P

Lactose-P is cleaved by P-β-gal to yield glucose and galactose-6-P, which

are further catabolized

The enzyme systems of lactose PTS and P-β-gal are generally inducible,

and repressed by glucose

An equally common way for LAB to metabolise lactose is by means of a

permease and subsequent cleavage by β-gal to yield glucose and galactose,

which may again enter the major pathways

Fructose 1,6-P

ATP

ADP

NADH

+ P i

Glucose-6-P

Fructose-6-PATP

ADP

Dihydroxyacetone-P Glycerinaldehyde3-P

1,3-Biphosphoglycerate

ATP

NAD +

ADP

3-Phosphoglycerate

2-Phosphoglycerate

PhosphoenolpyruvateH 2 O

ATP

Glucose

Pyruvate

ADP

Lactose Lactose Lactose

PEP/PTS Permease Permease

Lactose-P

Galactose-6-P

Tagatose-6-P

Tagatose-1,6-P

ATP

ADP

Lactose

Galactose

Galactose-1-P

ATP

ADP

Glucose-1-P

Lactose

Glucose-6-P

GlucoseATP

ADP

NADP +

NADPH

6-Phosphogluconate

CO 2Ribulose-5-P

Xylulose-5-P

Acetyl-P

CoASH

P i

Acetyl-CoANADH

NADAcetaldehyde

NADH

NADEthanol

CoASH

NADHNAD +

Lactate

Acetate

P i

IN

OUT

NADP + NADPH

Biochemistry of the Fermentation Process

Citrate is present in milk (9 mM) and can also serve as energy source

for LAB

Next to lactose, citrate metabolism plays an important role in milk

fermentations

The ability of LAB to metabolize citrate is invariably linked to

endogenous plasmid that contains the gene encoding the transporter,

which is responsible for citrate uptake from the medium

COOH

C

CH2

HOOC OH

COOH

COOH

CH3CH2

HOOC

COOH

C O

CH3

C O

COOH

CH3

HC OH

COOH

2X acetate

+

2X oxaloacetate

2X citrate

2X lactate

2X CO2

1

2

3

2X pyruvate

2X NAD2X NADH

Citrate catabolism

CH3

C O

COOH

2X pyruvate

4 CO2

CH3

C OH

COOH

a-acetolactate

CO2

diacetyl

H3C

C

O

CH3H3C

C

O

C

O

acetoin

CH3H3C

C

O

CH

OH

CO25

6

NADH NAD

2,3-butandiol

CH3H3C

CH

OH

CH

OH

6

NADH

NAD

CH 3CO-CoA7

8

2X CO 22X NADH 2X NAD

2X formate

HCOOH

2X acetyl-CoA

CH3

CHO

2X acetaldehyde

10

2X NADH

2X NAD

CH3

CH2OH

2X ethanol

2X NADH

2X NAD

10

9

CH 3COOH

2X acetate

2X ADP

2X ATP

Citrate catabolism

Biochemistry of the Fermentation Process

Products of citrate catabolism, such as diacetyl, acetaldehyde and acetoin, have distinct aroma properties and influence significantly the quality of fermented foods

For instance, diacetyl determines the aromatic properties of fresh cheese, fermented milk, cream and butter

The production of CO2 can add to the texture of some fermented dairy products

Not all LAB are able to metabolize citrate

Lactococcus lactis, Leuconostoc sp. and Enterococcus have been studied

Biochemistry of the Fermentation Process

Proteolysis is the most complex event during cheese ripening

It is important for flavour and texture development

The main proteolytic agents in cheese manufacture are

- the rennet

- the indigenous milk protease (plasmin) and

- the proteolytic enzymes of the microflora

Biochemistry of the Fermentation Process

Biochemistry of the Fermentation Process

2HN…................................................................ Phe105 — Met106 …........COOH

ρεννίνη

γλυκομακροπεπτίδιοπαρα-κ-καζεΐνη

2HN…...........................................................Phe105 Met106........COOH

Biochemistry of the Fermentation Process

Gastric proteinases from calves, kids and lambs have been used

traditionally as rennet

Plant proteinases (extracts from dried flowers of Cynara cardunculus)

have been employed successfully for many centuries in Portugal and in

Spain

They are claimed to be similar to chymosin in terms of specificity and

kinetic parameters, but they produce higher levels of the ripening index

than commercial animal rennet

Biochemistry of the Fermentation Process

Plasmin is the indigenous milk proteinase, and it is a trypsin-like serine

proteinase

Its complex consists of the active enzyme (plasmin), its zymogen

(plasminogen), plamsinogen activators and enzyme inhibitors

Activators are associated with the casein micelles, while the inhibitors are

in the serum phase

Plasmin is active on all caseins, especially αs2- and β-, but it shows very low

activity on κ-casein

Biochemistry of the Fermentation Process

Plasmin activity is cheese is clearly indicated by the formation of γ-caseins

from β-casein during ripening

Pasteurization increases plasmin activity in milk, probably because of the

inactivation of plasmin inhibitors and by increasing the rate of plasminogen

activation

In hard cooked cheeses like Swiss type cheeses or the Italian “grana”

cheeses, plasmin is mainly responsible for the primary hydrolysis of caseins,

since rennet is almost inactivated during cooking

Biochemistry of the Fermentation Process

LAB have limited abilities to synthesize amino acids

Milk contains insufficient amounts of free amino acids to sustain LAB

growth

Although weak proteolytic bacteria, LAB posses a complex proteolytic

system capable of hydrolysing milk proteins to peptides and amino acids

Their proteolytic system contributes to the degradation of milk protein

and hence to the texture, taste and aroma of dairy fermented products

oligopeptidetransport system

di/tripeptidetransport system

aminoacidstransport system

largeoligopeptides

smalloligopeptides

di/tripeptides

amino acids

peptidases

peptidases

peptidases

WALL MEMBRANE CYTOPLASM

proteinase

oligopeptides

di/tripeptides

amino acids

Biochemistry of the Fermentation Process

A number of enzymes are involved in the conversion of amino acids to

flavour components

These enzymes may catalyse reactions such as deamination, transamination,

decarboxylation, and cleavage of the amino acid side chain

Branched-chain amino acids can be transaminated to keto acids,

which then undergo either spontaneous degradation or

they are enzymatically converted to

the corresponding aldehydes or carboxylic acids

amino acids

decarboxylation

CO 2

amines

oxidative deamination

NH 3 aldehydes

transaminationoxidative deamination

amino acidsalpha-ketoacids

NH 3degradation

phenols

sulfur compounds

indoles

reduction oxidation

alcohols acids

Biochemistry of the Fermentation Process

Enzymes involved

AT aminotransferaseGDH glutamate dehydrogenaseHADH hydroxy acid dehydrogenaseKADH keto acid dehydrogenaseKADC keto acid decarboxylaseAlcDH alcohol dehydrogenaseAldDH aldehyde dehydrogenase

Compound formed

Type Formula Phe Leu Met

a-keto acid R-CH2-CO-COOH Phenylpyruvate a-ketoisocaproate a-ketomethylthiobutyrate

Hydroxy acid R-CH2-CHO-COOH Phenyllactate Hydroxyisocaproate Hydroxymethylthiobutyrate

Carboxylic acid R-CH2-COOH Phenylacetic acid Isovaleric acid 3-Methylthiopropionic acid

Aldehyde R-CH2-CHO Phenylacetaldehyde 3-Methylbutanal orisovaleraldehyde

Methional

Alcohol R-CH2-CHOH 3-Methylbutanol Methional

Other volatile S-compounds

Methanethiol, dimethyl disulfide, dimethyl trisulfide

Compounds resulting from Phe, Leu and Met degradation

diacetyl

butter flavor

Butter

isovalerate

isobutyrate

sweaty notes

phenylacetate

phenylacetaldehyde

fruity notes

Emmental

3-methyl-butanal

spicy cocoa flavor

Parmesan

CH3CH(CH3)CH2CHO

Biochemistry of the Fermentation Process

3-methyl butanal, originating from leucine has been characterised as an

important volatile compound formed during the ripening of Parmesan cheese,

which is responsible for a spicy cocoa flavour

Most of these activities have been demonstrated in vitro for LAB,

but how and whether they proceed in vivo is not always easy to establish

Biogenic amines

Biochemistry of the Fermentation Process

Biochemistry of the Fermentation Process

Bioactive peptides present in the amino acid sequence of milk proteins

especially from β-casein (β-casomorphins)

Although other animal as well as plant proteins contain potential bioactive

sequences, milk proteins are currently the main source of a range of

biologically active peptides

The structures of biologically active sequences were obtained from in vitro

enzymatically and /or by in vivo gastrointestinal digest of the appropriate

precursor proteins

Some known casomorphins

β-Casomorphin 1-3 H-Tyr-Pro-Phe-OH

β-Casomorphin 1-4 H-Tyr-Pro-Phe-Pro-OH

β-Casomorphin 1-4, amide H-Tyr-Pro-Phe-Pro-NH2

β-Casomorphin 5 H-Tyr-Pro-Phe-Pro-Gly-OH

β-Casomorphin 7 H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH

β-Casomorphin 8 H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-OH

Biochemistry of the Fermentation Process

Bioactive peptides are potential modulators of various regulatory processesin human body

- Opioid peptides are

opioid receptor ligands,

which can modulate absorption processes in the intestinal tract

- Angiotensin-I-converting enzyme (ACE)-inhibitory peptides are

hemodynamic regulators and exert an antihypertensive effect

Biochemistry of the Fermentation Process

- Immunomodulating casein peptides

stimulate the activities of cells of the immune system

- Antimicrobial peptides

kill sensitive microorganisms

- Antithrombotic peptides

inhibit aggregation of platelets

caseinophosphopeptides may function as carriers

for different minerals, especially Ca

Biochemistry of the Fermentation Process

The main lipolytic agents in cheese include the

- indigenous milk lipoprotein lipase

- lipases and esterases produced by

the starter and non-starter bacteria

and depending on the cheese variety

- enzyme preparations added during manufacturing

Biochemistry of the Fermentation Process

Milk lipase causes significant lipolysis in raw milk cheese

It is inactivated during pasteurisation at 76oC for 10 sec

It is selective for fatty acids at the sn-3 position, where the most

of the butyric acid in milk fat is esterified

In most cheese varieties relatively little lipolysis occurs, with the

exception of mould-ripened cheeses

Biochemistry of the Fermentation Process

LAB are generally considered as low lipolytic

LAB lipolytic system has not been extensively studied

compared to the proteolytic system,

although there are several reports on lipases and esterases of LAB

Vital information on

their cellular location of all these enzymes,

their specificity as well as

their overall role during ripening

remains to be determined

Production of EPS by LAB

Generalized diagram of the conversion of lactose, galactose

and glucose to EPS

Improve rheology & texture of food

Enhance perception of taste

Enhance colonization of LAB in GI tract

Immunostimulatory effects

Antitumor effects

Lower blood cholesterol

Biochemistry of the Fermentation Process

The bacteriocins produced by LAB are antimicrobial peptides

- ribosomally synthesised

- small

- cationic

- amphiphilic (rather hydrophobic)

which vary in

- spectrum of activity

- molecular structure

- molecular mass

- thermostability

- pH range of activity

Biochemistry of the Fermentation Process

Class I bacteriocins = lantibiotics

small, cationic, hydrophobic, and heat-stable peptides

contain unusual amino acids (lanthionine and/or 3-methyl-lanthionine)

Class Ia lantibiotics

cationic and hydrophobic peptides

with flexible, linear structure

act by forming pores in target membranes

(e.g. nisin produced by several strains of Lactococcus lactis )

Class Ib lantibiotics

rigid globular peptides with no net charge or a net negative charge

(e.g. mersacidin produced by Bacillus subtilis)

Schematic representation of the structure of the lantibiotic nisin A

Dehydrations of serines

and threonines

yield 2,3-didehydroalanines (Dha)

and 2,3-didehydrobutyrines (Dhb),

respectively

The formation of thioetherbonds

between cysteines

and dehydrated residues results in

lanthionine (Lan: Ala-S-Ala)

and

3-methyllanthionine (MeLan: Abu-S-Ala, in which Abu is an

aminobutyric acid)

Prepeptide maturation and primary structure of lantibiotics

Prepeptide maturation and primary structure of lantibiotics

Dehydrations of the boxed serines and threonines yield 2,3-didehydroalanines (Dha) and 2,3-didehydrobutyrines (Dhb), respectively

The formation of thioether bonds between cysteines and dehydrated residues results in lanthionine(Lan: Ala-S-Ala) and 3-methyllanthionine (MeLan: Abu-S-Ala, in which Abu is an aminobutyric acid)

Gene clusters involved in production of, and immunity to, lantibiotics

Schematic representation of nisin biosynthesis and regulation in Lactococcus lactis

Hijacking of lipid II for pore formation

(a) The structure of lipid II is composed of an N-acetylglucosamine-b-1,4-N-acetylmuramic acid disaccharide (GlcNAc–MurNAc), attached by a pyrophosphate to a membrane anchor of 11 isoprene units (undecaprenylpyrophosphate). A pentapeptide is attached to the muramic acid. Transpeptidase and transglycosylase enzymes cross-link the disaccharide and pentapeptidegroups of multiple lipid II molecules to generate the peptidoglycan

(b) The amino-terminus of nisin binds lipid II while the carboxy-terminus inserts into the bacterial membrane. Four lipid II and eight nisinmolecules have been shown to comprise a stable pore, although the arrangement of the molecules within each pore is currently unknown

(c) The NMR solution structure of the 1:1 complex of nisin and a lipid II derivative in DMSO. Nisinis illustrated in red, the prenyl chain of lipid II is depicted in white, the phosphate atoms of the pyrophosphate group are shown in orange, and the muramic acid is portrayed in blue. The N-acetylglucosamine (GlcNAc) is shown in magenta and the pentapeptide chain is presented in yellow.

Biochemistry of the Fermentation Process

Class II bacteriocins

small, cationic, hydrophobic, heat-stable peptides

that are not post-translationally modified

Class IIa pediocin-like bacteriocins with strong antilisterial effect

Class IIb two-peptide bacteriocins

Class IIc bacteriocins that do not belong to the other subgroups

Class III bacteriocins

large, hydrophilic, heat-labile proteins

Leader peptide propeptide

macedocin -MEKETTIIESIQEVSLEELDQIIGA --GKNGVFKTISHECHLNTWAFLATCCS

Streptococcin A-M49 -MTKEHEIINSIQEVSLEELDQIIGA --GKNGVFKTISHECHLNTWAFLATCCS

Streptococcin A-FF22 -MEKNNEVINSIQEVSLEELDQIIGA --GKNGVFKTISHECHLNTWAFLATCCS

Lacticin 481 --MKEQNSFNLLQEVTESELDLILGA -KGGSGVIHTISHECNMNSWQFVFTCCS

Butyrivibriocin OR79 ---MNKELNALTNPIDEKELEQILGG ---GNGVIKTISHECHMNTWQFIFTCCS

Variacin ----MTNAFQALDEVTDAELDAILG- --GGSGVIPTISHECHMNSFQFVFTCCS

Mutacin II MNKLNSNAVVSLNEVSDSELDTILGG NRWWQGVVPTVSYECRMNSWQHVFTCC-

Macedocin, the lantibiotic produced by S. macedonicus

K R tnp A A’ A1 M T F E

mac cluster

Regulation Biosynthetic Immunity

K R A A1 M T F E G tnpA partial

scncluster

Inhibitory spectrum

of macedocin

0

20

40

60

80

100

120

Bacil

lus sp

.

Brocho

nthrix

sp.

Clostr

idium

sp.

Lister

ia sp.

Staph.

aureu

s

Induction Factor

of macedocin biosynthesis

Macedocin is produced only when

S. macedonicus is grown in milk !

Peptides

of low molecular mass

corresponding to

fragments of

β-lactoglobulin

αS1-casein

β-casein

Macedocin, the lantibiotic produced by S. macedonicus

Inhibitory spectrum of macedocin

Indicator organismMacedocin activity

(mm inhibition zone)

S. agalactiae LMG 14694T 8

S. anginosus LMG 14502T 9

S. bovis LMG 8518T 7

S. equinus LMG 14897T 10

S. gordonii LMG 14518T 10

S. mutans LMG 14558T 0

S. oralis LMG 14532T 10

S. pneumoniae LMG 14545T 13

S. pyogenes LMG 21599T 13

S. salivarius LMG 11489T 7

S. sobrinus LMG 14641T 0

S. thermophilus LMG 6896T 15

Lactococcus lactis ACA-DC 6890T 15

Species-specific PCR of S. macedonicus coloniesisolated throughout ripening of Cheese C

1: B1 (unripened Baski) 2: B2 (ripened Baski)

3: K0 (Kasseri d0) 4: K7 (Kasseri d7)

5: K15 (Kasseri d15) 6: K30 (Kasseri d30)

7: K60 (Kasseri d60) 8: K90 (Kasseri d90)

9: positive control 10: blank

Sample

Mpi B1 B2 K0 K7 K15 K30 K60 K90

log

cfu/

gr

3

4

5

6

7

8

9

Enumeration of S. macedonicus

on M17 agar, supplemented with

25 μg/ml streptomycin, throughout ripening of Cheese C

S. macedonicus in Kasseri cheese

Macedocin detection in Kasseri cheese during ripening

A

B

1 2 3 4

5 6 7 8

1 2 3 4

5 6 7 8

C

1: B1 2: B2 3: K0 4: K7

5: K15 6: K30 7: K60 8: K90

In the center the negative control (Cheese A)

Cheese samples heated at 80οC

for 10 min

Cheese extracts

Sample

Mi B1 B2 K0 K10 K30 K60 K90 K120

Log

MPN

/ml

-1

0

1

2

3

4

Sample

Mi B1 B2 K0 K10 K30 K60 K90 K120

log

MPN

/ml

-1

0

1

2

3

4

5

Inhibition of Clostridium tyrobutyricum by S. macedonicus ACA-DC

198 under conditions simulating Kasseri cheese production

ClostridiumSpores

ClostridiumVegetative cells

Inhibition of Bacillus cereus in bread prepared from sourdough containing Lactobacillus sanfranciscensis

Lactic acid bacteria in sourdough

Lactic acid bacteria in wine

Malic acid Lactic acid

Oenococcus oeni

- CO2

The malolactic fermentation in wine

- Decreases the acidity of the wine and

- Influences the flavor of wine

Effect of L. plantarum on Fusarium

during malting of barley

Effect of L. plantarumon mash filterability

Lactic acid bacteria in beer